The proposed research involves defining the detailed molecular mechanisms by which L-triiodothyronine (T3 affects the metabolism of calcium ion (Ca++) within thyrotropic cells so as to determine a mechanism of T3 inhibition of thyrotropin (TSH) secretion. A cell culture system comprised only of TSH-producing cells derived from mouse pituitary thyrotropic tumors (TtT cells) has been developed which is responsive to physiological concentrations of thyroid hormones. We have demonstrated a critical role for Ca++ in the regulation of TSH secretion from TtT cells which conforms to the more general scheme of stimulation-secretion coupling as originally conceived by Douglas. Thyrotropin releasing hormone (TRH) has been shown to stimulate radiocalcium (45Ca++ uptake by TtT cells and to enhance the rate of 45Ca++ efflux from pre-loaded cells. Preincubation of TtT cells with 10 nM T3 virtually abolished TRH-stimulated 45Ca++ uptake. In TtT cells we will study the effects of T3 on Ca++ fluxes and concentrations and on the intracellular processes at the plasma membrane which serve to control the free or unbound concentration of Ca++ in the cytosol. Ca++ fluxes will be monitored with 45Ca++ and stable Ca++ concentrations measured by a method we have developed using the metallochromic indicator, arsenazo III. Changes in the concentration of free cytosolic Ca++ will be monitored using the Ca++ sensitive photoprotein aequorin after direct intracellular injection. The activity of the Ca++ pump will be measured as Ca++-ATPase activity and by its ability to transport Ca++ against the concentration gradient. Phospholipid metabolism will be measured by (32P) orthophosphate incorporation and calmodulin concentrations by a specific radioimmunoassay. The activity of the "Na pump" will be measured as Na+-K+ ATPase activity and by fluxes of 22 Na+ and 86Rb+, the number of pump units by (3H)ouabain binding and the membrane polarization measured indireectly using a lipophilic cation, triphenylmethylphosphonium ion. The effects of T3 on these various processes will be integrated so as to define the regulation of intracellular Ca++ metabolism by T3 and the role of Ca++ as a critical intracellular regulator of TSH secretion. These studies are part of the long-term objective of defining the molecular mechanisms that regulate TSH secretion.